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Vibrio harveyi, which belongs to family Vibrionaceae of class Gammaproteobacteria, includes the species V. carchariae and V. trachuri as its junior synonyms. The organism is a well-recognized and serious bacterial pathogen of marine fish and invertebrates, including penaeid shrimp, in aquaculture. Diseased fish may exhibit a range of lesions, including eye lesions/blindness, gastro-enteritis, muscle necrosis, skin ulcers, and tail rot disease. In shrimp, V. harveyi is regarded as the etiological agent of luminous vibriosis in which affected animals glow in the dark. There is a second condition of shrimp known as Bolitas negricans where the digestive tract is filled with spheres of sloughed-off tissue. It is recognized that the pathogenicity mechanisms of V. harveyi may be different in fish and penaeid shrimp. In shrimp, the pathogenicity mechanisms involved the endotoxin lipopolysaccharide, and extracellular proteases, and interaction with bacteriophages. In fish, the pathogenicity mechanisms involved extracellular hemolysin (encoded by duplicate hemolysin genes), which was identified as a phospholipase B and could inactivate fish cells by apoptosis, via the caspase activation pathway. V. harveyi may enter the so-called viable but nonculturable (VBNC) state, and resuscitation of the VBNC cells may be an important reason for vibriosis outbreaks in aquaculture. Disease control measures center on dietary supplements (including probiotics), nonspecific immunostimulants, and vaccines and to a lesser extent antibiotics and other antimicrobial compounds.

Culturing has been the cornerstone of microbiology since Robert Koch first successfully cultured bacteria in the late nineteenth century. However, even today, the majority of microorganisms in the marine environment remain uncultivated. There are various explanations for the inability to culture bacteria in the laboratory, including lack of essential nutrients, osmotic support or incubation conditions, low growth rate, development of micro-colonies, and the presence of senescent or viable but nonculturable (VBNC) cells. In the marine environment, many bacteria have been associated with dormancy, as typified by the VBNC state. VBNC refers to a state where bacteria are metabolically active, but are no longer culturable on routine growth media. It is apparently a unique survival strategy that has been adopted by many microorganisms in response to harsh environmental conditions and the bacterial cells in the VBNC state may regain culturability under favorable conditions. The resuscitation of VBNC cells may well be an important way to cultivate the otherwise uncultured microorganisms in marine environments. Many resuscitation stimuli that promote the restoration of culturability have so far been identified; these include sodium pyruvate, quorum sensing autoinducers, resuscitation-promoting factors Rpfs and YeaZ, and catalase. In this review, we focus on the issues associated with bacterial culturability, the diversity of bacteria entering the VBNC state, mechanisms of induction into the VBNC state, resuscitation factors of VBNC cells and implications of VBNC resuscitation stimuli for cultivating these otherwise uncultured microorganisms. Bringing important microorganisms into culture is still important in the era of high-throughput sequencing as their ecological functions in the marine environment can often only be known through isolation and cultivation.

Aeromonas salmonicida was isolated from red spot emperor, king soldier bream, white-spotted rabbit fish and tilapia, and an invertebrate (abalone) in Oman during December 2011–May 2012. The cytotoxic enterotoxin ast gene was found widely distributed among the isolates; aerolysin-like protein (act) and the flagellin structural gene lafA less so; and the nuclease gene (nuc) not at all. However, there was not any evidence of pathogenicity among the isolates when tested in laboratory-based experiments using rainbow trout and Nile tilapia. Therefore, the risk of the pathogen to fish in Oman is unclear.

Since the late nineteenth century, pure cultures have been regarded as the cornerstone of bacteriology. However, not all bacteria will multiply sufficiently to produce visible colonies on solid media; some cells will produce micro-colonies that are invisible to the naked eye. Moreover, the proportion of culturable cells that produce visible growth will vary according to the species and the state of the cells-are they actively growing or comparatively inactive? The latter have a poorer rate of recovery in terms of cultivability. It is unclear whether or not an individual colony is always derived from a single cell; it is possible that organisms in close proximity to each other may multiply and come together to produce single colonies. Then, the resultant growth will most certainly be derived from more than one initial cell. Although it is generally assumed that streaking and re-streaking on fresh media will purify any culture, there is evidence for microbial consortia interacting to form what appear to be single pure cultures. As so-called pure cultures underpin traditional microbiology, it is relevant to understand that the culture does not necessarily contain clones of identical bacteria, but that there may be variation in the genetic potential of the component cells, i.e. the cells are not homogeneous. Certainly, many bacteria change rapidly upon culturing, with some becoming bigger and less active. It is difficult to be sure if these changes reflect a loss or change of DNA or whether standard culturing methods select faster growing cells that are effectively not representative of the environment from which they were derived. These concepts are reviewed with an emphasis on bacterial fish pathogens.

This study characterized probiotics Kocuria SM1 and Rhodococcus SM2, which were recovered from the intestinal microbiota of rainbow trout (Oncorhynchus mykiss, Walbaum). The cultures were Gram-positive, non-motile, catalase-positive and oxidase-negative cocci or rods. Cell multiplication of SM1 and SM2 was observed at 4–37 °C (45 °C for SM1), in 0–20% (w/v) NaCl and at pH 2–11. The viability was not affected when exposed to pepsin at pH 2.0 and 3.0, and pancreatin at pH 8.0. Neither isolates were chrome azurol S-positive for siderophore production. Of the 19 common enzymes analysed using the API-ZYM system, only 8 were evident in the culture of SM1 compared to 11 enzymes for SM2. The secondary metabolites of both probiotics were inhibitory to Acinetobacter baumannii, Vibrio anguillarum and V. ordalii; SM2 inhibited Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. SM2 was resistant to penicillin and sulphatriad, out of six antimicrobial agents; SM1 was resistant to sulphatriad. These results suggest that Kocuria SM1 and Rhodococcus SM2 are able to grow over a wide range of temperature, salinity and pH, including in conditions that mimic the gastrointestinal environment of fish and produce extracellular enzymes that may have a role in the host digestive processes. Importantly, Rhodococcus SM2 displays a high degree of bacteriocinogenic potential against multi-drug-resistant human pathogens that have never been documented among the gut microbiota of fish.

During a 3-month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.

International audience; 150 scientists from 26 countries assembled in the John McIntyre Centre at the University of Edinburgh from April 1–4 2014 for VIBRIO2014, a conference dedicated to the study of the biology and biotechnology of vibrios. These Gram-negative aquatic bacteria are widespread in the estuarine and coastal marine environments. They play an important role in marine ecology, e.g. petroleum degradation, and they are also associated with human [e.g. Vibrio cholerae the causal agent of cholera] and animal diseases [e.g. Vibrio anguillarum the cause of vibriosis in fish). In total, there were 54 oral and 72 poster presentations grouped in 7 sessions. After the official opening by the Scottish Minister for Environment and Climate Change, Paul Wheelhouse MSP, Rita Colwell delivered the keynote address, entitled: “Vibrios past, present and future”.

The effects of dietary probiotics on growth performance, health status of the digestive system and on the blood parameters of juvenile rainbow trout ( Oncorhynchus mykiss , Walbaum) were investigated. Compared to the controls, diets supplemented with Kocuria SM1 and Rhodococcus SM2 for 14 days caused no significant (P > 0.05) change on the growth of fish, although SM2 led to an increased specific growth rate. SM1 or SM2 started to colonize in the gut after 7 days of administration reaching a maximum, i.e. ~ 90–100% of total bacterial population, at 14 days. Yet, the probiotics disappeared from the gut once administration ceased. Structural differences were not noted in the pyloric caeca and foregut, but the overall condition of the midgut and hindgut improved in fish receiving probiotics. The profile of digestive enzymes, as determined by the use of API ZYM, and stress indicators, i.e. serum hemoglobin, urea, creatinine and glucose, was not affected by any of the experimental diets (P > 0.05).

A study was made of the biochemical, cultural, morphological, physiological and serological characters of 25 Gram-positive bacterial isolates of bacterial kidney disease in salmonid fish. Two distinct homogenous phena and seven single-member clusters were defined as a result of overall similarity based on analyses with the Jaccard coefficient. One phenon was equated withCorynebacterium pyogenes, but the second represents a novel taxon.

The taxonomic position of strain DFH11(T), which was isolated from coastal seawater off Qingdao, People's Republic of China in 2007, was determined. Strain DFH11(T) comprised Gram-negative, motile, strictly aerobic spirilli that did not produce catalase. Comparative 16S rRNA gene sequence analysis revealed that strain DFH11(T) shared ~97.2, 93.3, 91.8, 91.7 and 91.5% sequence similarities with Oleispira antarctica, Spongiispira norvegica, Bermanella marisrubri, Oceaniserpentilla haliotis and Reinekea aestuarii, respectively. DNA-DNA hybridization experiments indicated that the strain was distinct from its closest phylogenetic neighbour, O. antarctica. The strain grew optimally in 2-3% (w/v) NaCl, at pH 5.0-10.0 (optimally at pH 7.0) and between 0 and 30°C (optimum growth temperature 28°C). The strain exhibited a restricted substrate profile, with a preference for aliphatic hydrocarbons, that is consistent with its closest phylogenetic neighbour O. antarctica. Growth of the isolate at different temperatures affected the cellular fatty acid profile. 28°C cultured cells contained C(16:1)ω7c and/or iso-C(15:0) 2-OH (50.4%) and C(16:0) (19.2%) as the major fatty acids. However, the major fatty acids of the cells cultured at 4°C were C(16:1)ω7c and/or C(16:1)ω6c (40.2%), C(16:0) (17.2%) and C(17:1)ω8c (10.1%). The G+C content of the genomic DNA was 42.7 mol%. Phylogeny based on 16S rRNA gene sequences together with data from DNA-DNA hybridization, phenotypic and chemotaxonomic characterization revealed that DFH11(T) should be classified as a novel species of the genus Oleispira, for which the name Oleispira lenta sp. nov. is proposed, with the type strain DFH11(T) (=NCIMB 14529(T) = LMG 24829(T)).

Aims: The study investigated antigen characteristics of biotype (bt) 1 and bt 2 isolates of Yersinia ruckeri. Methods and Results: The cell surface characteristics of Y. ruckeri were compared for their antigenic characteristics using polyclonal antibodies that revealed that both biotypes had a homogenous whole-cell protein antigenic profile. Notable differences in the antigenic properties were observed in the lipopolysaccharide profile of both biotypes. Two iron-regulated outer membrane proteins (IROMP) of c. 90 and 100 kDa were shown to be major specific antigens. The results demonstrate for the first time differences in antigens between bt 1 and bt 2 isolates of serotype O1 isolates of Y. ruckeri. The protection induced in rainbow trout by a commercial monovalent, and bivalent inactivated vaccine was tested with the outcome that the ability of isolates to cause mortality in vaccinated fish varied with geographical location. In this context, vaccination studies suggested that the O antigen was the dominant immunogenic molecule involved in protection against the disease. Conclusions: The O antigen of Y. ruckeri was the dominant immunogenic molecule involved in the protection of rainbow trout against enteric redmouth disease. Significance and Impact of the Study: There are distinct phenotypic and antigenic differences in Y. ruckeri bt 1 and bt 2 with O antigen recognized as the dominant immunogenic molecule. The data have significance in explaining the lack of success of the earlier monovalent vaccine and demonstrate the effectiveness of the newer bivalent vaccine.

The biochemical and cell surface characteristics of 63 non-motile isolates of Yersinia ruckeri from various sources were compared using the API 20E rapid identification system and conventional phenotypic methods. Eight individual phenotypic groups from a variety of fish species were observed from the data set. Non-motile isolates were not exclusively observed from serogroup O1; membership of biotype 2 was recorded for representatives from serogroups O2-O7. Variations in phenotypes highlights that new clonal groups are arising and that the current typing scheme requires expansion. Previously, it was hypothesized that disease was caused by a few virulent clones; data in this paper suggests that this assumption is not the case. The lipopolysaccharide (O antigen) type in the non-motile biotype was different from other isolates of Y. ruckeri.

Edwardsiella tarda is a causative agent of edwardsiellosis in freshwater and marine fish. Extracellular enzymic, haemolytic, hydrophobic and serum resistance activities, haemagglutination, autoagglutination and siderophores of high- and low- virulent E. tarda strains were examined. The results revealed different haemagglutination, autoagglutination, haemolytic, hydrophobic and serum resistance activities in different strains. Analysis of extracellular proteins (ECPs) and outer membrane proteins (OMPs) demonstrated several major, low molecular weight, virulent-strain-specific proteins, which could be virulence-related. Based on the database search with MALDI-TOF MS data, the closest homologies of the three protein bands Ed1, Ed2 and Ed3 were phosphotransferase enzyme family protein, nitrite reductase [NAD(P)H], large subunit and ATP-dependent Lon protease, respectively. A comparison of pathogenicity of purified lipopolysaccharide (LPS) and lipid A from virulent and avirulent strains demonstrated that LPS was one of the virulence factors of the E. tarda isolates, and lipid A was a biologically active determinant of LPS.

A taxonomic study was performed on strain D7015T, which was isolated from coastal sediment close to a coal-fired power station in Qingdao, China. Cells of strain D7015T were Gram-positive, non-motile, diphtheroid rods that grew in the presence of 0–8 % (w/v) NaCl and at 4–37 °C, with optimum growth at 1 % (w/v) NaCl and 30–32 °C. The DNA G+C content was 65.0 mol%. The major fatty acids were C18 : 1 ω9c (56.18 %), C16 : 0 (38.02 %), C16 : 1 ω7c (4.45 %), C18 : 0 (1.0 %) and C14 : 0 (0.35 %). On the basis of morphological, physiological and phylogenetic characteristics, strain D7015T was classified in the genus Corynebacterium. It exhibited a 16S rRNA gene sequence similarity of 95.9 % and a DNA–DNA relatedness value of 20.4 % with Corynebacterium halotolerans DSM 44683T. Strain D7015T was sufficiently different from recognized species of the genus Corynebacterium to be considered to represent a novel species. The name Corynebacterium marinum sp. nov. is proposed, with strain D7015T (=CGMCC 1.6998T=NRRL B-24779T) as the type strain.

As a result of the continuous evolution of microbial pathogens towards antibiotic-resistance, there have been demands for the development of new and effective antimicrobial compounds. Since the 1960s, the scientific literature has accumulated many publications about novel pharmaceutical compounds produced by a diverse range of marine bacteria. Indeed, marine micro-organisms continue to be a productive and successful focus for natural products research, with many newly isolated compounds possessing potentially valuable pharmacological activities. In this regard, the marine environment will undoubtedly prove to be an increasingly important source of novel antimicrobial metabolites, and selective or targeted approaches are already enabling the recovery of a significant number of antibiotic-producing micro-organisms. The aim of this review is to consider advances made in the discovery of new secondary metabolites derived from marine bacteria, and in particular those effective against the so called “superbugs”, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), which are largely responsible for the increase in numbers of hospital acquired, i.e., nosocomial, infections.

Aims: To determine the effect of lipopolysaccharide (LPS) for the prevention of infection by Aeromonas hydrophila in rainbow trout (Oncorhynchus mykiss Walbaum) fingerlings. Methods and Results: Rainbow trout fingerlings were fed with 0 mg (= controls), 1·875 mg, 3·75 mg, 7·5 mg and 15 mg of LPS per 100 g of commercial feed for 14 days before experimental challenge with A. hydrophila. The results revealed a reduction in mortalities to 5% in the two lowest doses and 15% in the group, which received 15 mg LPS per 100 g of feed, compared with 45% mortalities in the control. LPS exerted a powerful oxidative burst effect and was a potent mediator of phagocytic, lysozyme, bactericidal and antiprotease activities and total protein. However, whereas there were increases in specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) in LPS-treated fish, the data were not significantly (P > 0·05) different. Conclusions: LPS was effective at preventing disease caused by A. hydrophila and in stimulating the innate immune response of rainbow trout. Significance and Impact of the Study: The results of this study highlight the role of LPS in fish disease control.

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss, conferred protection against a new biogroup of Yersinia ruckeri. Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80-100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with 'Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.

Ginger, Zingiber officinale, which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila. At 0.5 g ginger per 100 g of feed, there was a reduction in mortalities to 0% compared with the controls (64%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was proliferation in the number of neutrophils, macrophages and lymphocytes, and enhanced phagocytic, respiratory burst, lysozyme, bactericidal and anti-protease activities compared with the controls.

Aims: The aim of this study was to assess the efficacy of in-feed probiotics as a preventive measure against skin infections caused by Aeromonas bestiarum and Ichthyophthirius multifiliis (Ich) in rainbow trout. Methods and Results: Fin rot was induced in fish by intradermal injection with 0·1 ml volumes containing 105 cells per ml A. bestiarum at the base of the dorsal fin. Ich infections resulted from immersion in Ich-contaminated water. Each probiotic was administered orally [108 cells per g feed for GC2 (Aeromonas sobria) and 1010 cells per g feed for BA211 (Brochothrix thermosphacta)] for 14 days. Results showed that, after challenge with A. bestiarum, probiotics GC2 and BA211 led to 76% and 88% survival, respectively, in contrast to 22% survival for controls. Fish fed with probiotic GC2 had 100% survival after challenge with Ich compared with 2% for probiotic BA211 and 0% for controls. Analysis of innate immune responses revealed that probiotic GC2 promoted higher phagocytic activity, whereas probiotic BA211 led to enhanced respiratory burst activity. Conclusion: Of the two probiotics examined, GC2 was more effective in protecting against both fin rot and Ich. Each probiotic appeared to stimulate different pathways within the innate immune system. Significance and Impact of the Study: This is the first demonstration that probiotics can protect fish against surface infections. Furthermore, this is the first time a probiotic has been shown to protect against a eucaryotic pathogen, namely I. multifiliis.

An increasing number of Gram-negative chromogens have become associated with fish diseases particularly involving the gills and external surfaces [fin/tail rot]. New additions to the list of pathogens include Chryseobacterium piscicola, Flavobacterium oncorhynchi, Tenacibaculum dicentrarchi, T. discolor, T. gallaicum and T. soleae with research interest focusing on molecular diagnoses and disease control by vaccination, probiotics, immunostimulants and bacteriophages.

The scientific literature abounds with references to aeromonads as fish pathogens, and it is often difficult to distinguish the primary pathogen from opportunist or contaminant. There has been a tendency for motile aeromonads to be linked with Aer. hydrophila and non motile representatives with Aer. salmonicida. With the advent of more reliable taxonomic methods, the reliability of some of the earlier identifications may be questioned with the results that more species of fish pathogenic aeromonads are being recognised. Thus, there is evidence that Aer. bestiarum, Aer. hydrophila subsp. dhakensis (haemorrhaging and generalized liquefaction; Fig. 4.1), Aer. sobria biovar sobria and Aer. veronii biovar sobria may pose problems to fish health (Orozova et al. 2009). Of these, Aer. hydrophila subsp. dhakensis has been reclassified in Aer. aquariorum (Martinez-Murcia et al. 2009). In addition, aeromonads are often encountered in diseased fish, although the proof of pathogenicity may be lacking. For example, Beaz-Hidalgo et al. (2010) recovered Aer. bestiarum, Aer. hydrophila, Aer. media, Aer. piscicola, Aer. salmonicida and Aer. sobria from diseased fish but without demonstrating actual pathogenicity. Aer. piscicola is a newly described species from diseased fish, albeit without evidence of actual pathogenicity (Beaz-Hidalgo et al. 2009). Whereas the aeromonads used to be classified in Vibrionaceae, a new family was established to accommodate them, i.e. the family Aeromonadaceae (Colwell et al. 1986).

JB-1 and GC2, which were equated with Bacillus sp. and Aeromonas sobria respectively, were recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss and ghost carp, Cyprinus sp. respectively, and demonstrated effectiveness as probiotics for the control of infections caused by Aeromonas salmonicida, Lactococcus garvieae, Streptococcus iniae, Vibrio anguillarum, Vibrio ordalii and Yersinia ruckeri. When administered to rainbow trout (average weight = 12 g) for 14 days in feed dosed at 2 × 108 cells g−1 of feed, JB-1 led to a reduction in mortalities to 0–13% after challenge with a range of bacterial pathogens compared to 80–100% mortalities of the controls. Similarly, use of GC2 reduced mortalities to 0–16% following the challenge compared to 80–100% mortalities of the controls. The mode of action reflected nutrition, production of inhibitory substances and stimulation of the innate immune responses. Specifically, JB-1 and especially GC2 were positive for siderophore and chitinase production, and increased lysozyme, phagocytic and respiratory burst activity.

Aim: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. Methods and Results: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a concentration of 107 cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and α1-antiprotease and lymphocyte populations. Conclusions: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. Significance and Impact of the Study: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response.

Aims: The aim of this study was to understand the microbial community of intestinal contents and mucosal layer in the intestine of rainbow trout by means of culture-dependent conventional and independent molecular techniques. Methods and Results: Forty-one culturable microbial phylotypes, and 39 sequences from 16S rRNA and two from 18S rRNA genes, were retrieved. Aeromonadaceae, Enterobacteriaceae and Pseudomonadaceae representatives were the dominant cultured bacteria. Genomic DNA isolated from intestinal contents and mucus was used to generate 104 random clones, which were grouped into 32 phylotypes at 99% minimum similarity, most of which were affiliated with Proteobacteria (>70% of the total). However, unlike library C (intestinal contents), the phyla Bacteroidetes and Fusobacteria were not found in intestinal mucus (library M), indicating that the microbiota in the gut mucus was different from that of the intestinal contents. Twelve sequences were retrieved from denaturing gradient gel electrophoresis analysis, and dominant bands were mostly related to Clostridium. Conclusions: Many novel sequences that have not been previously recognized as part of the intestinal flora of rainbow trout were retrieved. Significance and Impact of the Study: The fish gut harbours a larger bacterial diversity than previously recognized, and the diversity of gut mucus is different from that of intestinal contents.

Rainbow trout ( Oncorhynchus mykiss , Walbaum) were challenged intraperitoneally with a sublethal dose of Vibrio anguillarum VIB1 and allowed to recover. Then, after 7 days, naive fish, (designated as ‘bystander’ fish) which had never been exposed to the pathogen, were introduced to the same tank. These swam with the adapted (recovered) fish for 7 days before both groups and a control (never exposed directly to the pathogen or to recovered fish) group were exposed to a lethal dose of VIB1. Mortality records were 100% in the control group within 3 days, 47% in the adapted group and 60% in the unchallenged bystander group, which swam with the adapted group. In both the latter groups, the time to death of the non-surviving fish was attenuated. This inter-animal communication of signals has previously been documented for animals exposed to ionizing radiation. Assays of tissues from control, challenged and ‘bystander fish exposed to the pathogen showed that a signal as yet unidentified but similar to that seen in bystanders to irradiated fish was being produced. This signal caused a sharp and transient increase in intracellular calcium and a decrease in clonogenicity in a well-characterized reporter assay.

Vibrio harveyi VHH hemolysin is a putative pathogenicity factor in fish. In this study, the hemolysin gene vhhA was overexpressed in Escherichia coli , and the purified VHH was characterized with regard to pH and temperature profiles, phospholipase activity, cytotoxicity, pathogenicity to flounder, and the signal peptide.

Vibrio harveyi, which now includes Vibrio carchariae as a junior synonym, is a serious pathogen of marine fish and invertebrates, particularly penaeid shrimp. In fish, the diseases include vasculitis, gastro-enteritis and eye lesions. With shrimp, the pathogen is associated with luminous vibriosis and Bolitas negricans. Yet, the pathogenicity mechanisms are imprecisely understood, with likely mechanisms involving the ability to attach and form biofilms, quorum sensing, various extracellular products including proteases and haemolysins, lipopolysaccharide, and interaction with bacteriophage and bacteriocin-like substances.

The results of numerous studies indicate that fish possess bacterial populations on or in their skin, gills, digestive tract, and light-emitting organs. In addition, the internal organs (kidney, liver, and spleen) of healthy fish may contain bacteria, but there is debate about whether or not muscle is actually sterile. Using traditional culture-dependent techniques, the numbers and taxonomic composition of the bacterial populations generally reflect those of the surrounding water. More modern culture-independent approaches have permitted the recognition of previously uncultured bacteria. The role of the organisms includes the ability to degrade complex molecules (therefore exercising a potential benefit in nutrition), to produce vitamins and polymers, and to be responsible for the emission of light by the light-emitting organs of deep-sea fish. Taxa, including Pseudomonas, may contribute to spoilage by the production of histamines in fish tissue.

A vaccine comprising cells of Aeromonas bestiarum grown in tryptic soy broth and atypical A. salmonicida cells produced in iron-limited and iron-supplemented media protected goldfish Carassius auratus when administered by immersion (dosage ≈ 5 × 107 cells/mL for 60 s) followed after 28 d by an oral booster (dosage = 5 × 107 cells/g of feed), which was fed for 7 d so that each fish received about 1 g of vaccine-containing feed. After challenge by intramuscular injection of a virulent culture of atypical A. salmonicida, the relative percent survival (RPS) was more than 90%. The approach was more successful than using a commercial furunculosis vaccine with or without supplementation with A. bestiarum or atypical A. salmonicida cells. Moreover, a smooth derivative of the virulent rough culture of atypical A. salmonicida was less effective as a vaccine candidate, yielding an RPS of only 65%. Low antibody titers of 1:39–1:396 were found in the vaccinated fish. The vaccinated fish had a significantly hi...

Minimization of stochastic complexity (SC) was used as a method for classification of genotypic fingerprints. The method was applied to fluorescent amplified fragment length polymorphism (fAFLP) fingerprint patterns of 507 Vibrionaceae representatives. As the current BinClass implementation of the optimization algorithm for classification only works on binary vectors, the original fingerprints were discretized in a preliminary step using the sliding-window band-matching method, in order to maximally preserve the information content of the original band patterns. The novel classification generated using the BinClass software package was subjected to an in-depth comparison with a hierarchical classification of the same dataset, in order to acknowledge the applicability of the new classification method as a more objective algorithm for the classification of genotyping fingerprint patterns. Recent DNA–DNA hybridization and 16S rRNA gene sequence experiments proved that the classification based on SC-minimization forms separate clusters that contain the fAFLP patterns for all representatives of the species Enterovibrio norvegicus, Vibrio fortis, Vibrio diazotrophicus or Vibrio campbellii, while previous hierarchical cluster analysis had suggested more heterogeneity within the fAFLP patterns by splitting the representatives of the above-mentioned species into multiple distant clusters. As a result, the new classification methodology has highlighted some previously unseen relationships within the biodiversity of the family Vibrionaceae.

A . A . N . N E A M A T A L L A H , S . J . D E W A R A N D B . A U S T I N . 2003. Aims: The aim of this study was to improve the selective isolation of Listeria monocytogenes from smoked haddock fillets. Methods and Results: Listeria selective agar (LSA) ‐ Oxford formulation was supplemented with 25 l gm l )1 of colistin sulphate and 30 l gm l )1 of nalidixic acid. Inocula from four smoked haddock fillets produced colonies (approx. 2‐13 bacteria g )1 ), identified as L. monocytogenes, on LSA supplemented with antimicrobial compounds (MLSA 1 ). Moreover, there was only negligible evidence of bacteria which were not L. monocytogenes on MLSA. In contrast, LSA supported dense bacterial growth, which was not equated with L. monocytogenes. Significance and Impact of the Study: The modified medium permitted the recovery of L. monocytogenes from smoked haddock fillets and reduced the growth of contaminating bacteria.

There has been increasing interest in the use of probiotics in aquaculture to control fish diseases (e.g. Douillet Langdon 1994; Gildberg, Mikkelsen, Sandaker & Ringo 1997; Kennedy, Tucker, Neidic, Vermeer, Cooper, Jarrell & Sennett 1998; DeSchrijver & Ollevier 2000; Robertson, O'Dowd, Burrells, Williams & Austin 2000), and a diverse range of Gram-positive (such as Carnobacterium inhibens) (Robertson et al. 2000) and Gram-negative bacteria, including Vibrio alginolyticus (Garriques & Arevalo 1995), have been evaluated at various times. However, the possible use of probiotics to control diseases of ornamental fish has been neglected. Consequently, following previous success with live (Irianto & Austin 2002) and inactivated probiotics in salmonids (Irianto & Austin, in press), research has focused on the usefulness of inactivated bacterial preparations for the control of atypical Aeromonas salmonicida infections in goldfish, Carassius auratus (L.). Goldfish, of average 40-50 mm in length, were obtained from a commercial farm in England, and maintained in aerated re-circulating dechlorinated fresh water at 17 degrees C. Their health status was examined immediately upon arrival in the aquaria and at 1-2-week periods thereafter (after Austin & Austin 1989).

Probiotics, which are micro-organisms or their products with health benefit to the host, have found use in aquaculture as a means of disease control, supplementing or even in some cases replacing the use of antimicrobial compounds. A wide range of microalgae (Tetraselmis), yeasts (Debaryomyces, Phaffia and Saccharomyces) and Gram-positive (Bacillus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Streptococcus and Weissella) and Gram-negative bacteria (Aeromonas, Alteromonas, Photorhodobacterium, Pseudomonas and Vibrio) has been evaluated. However, the mode of action of the probiotics is rarely investigated, but possibilities include competitive exclusion, i.e. the probiotics actively inhibit the colonization of potential pathogens in the digestive tract by antibiosis or by competition for nutrients and/or space, alteration of microbial metabolism, and/or by the stimulation of host immunity. Probiotics may stimulate appetite and improve nutrition by the production of vitamins, detoxification of compounds in the diet, and by the breakdown of indigestible components. There is accumulating evidence that probiotics are effective at inhibiting a wide range of fish pathogens, but the reasons for the inhibitions are often unstated.

Vibriosis is an economically important disease of fish, marine invertebrates (particularly penaeid shrimps), and large marine mammals and is responsible for high mortality rates in aquaculture worldwide. Some Vibrio species are also responsible for zoonoses, whereas others are relatively nonpathogenic. Using 16S- and 23S-based PCR reactions, we obtained species-specific patterns and a 470-bp band, respectively. DNA sequences obtained on the 23S rRNA gene allowed us to identify species-specific probes for Vibrio parahaemolyticus, V. alginolyticus, V. anguillarum and for a cluster of taxonomically related species: V. carchariae/harveyi/campbelii. A phylogenetic tree based on the 23S sequences confirmed previous results obtained by Western blotting.

Aerobic heterotrophic bacteria were isolated from the intestinal contents of Atlantic salmon, Salmo salar, rainbow trout, Oncorhynchus mykiss, and turbot, Scophthalmus maximus, on tryptone soya agar and De Man Rogosa and Sharpe agar, of which 11 of 177 (6% of the total) of the isolates were antagonistic to Aeromonas salmonicida. Four of these cultures, which were identified tentatively as A. hydrophila, Vibrio fluvialis, Carnobacterium sp. and an unidentified Gram-positive coccus, were beneficial to fish when fed singly or as an equi-mixture. Feed supplemented with the putative probiotics indicated survival of the organisms in the gastrointestinal tract for 7 days. Feeding with the probiotics for 7 and 14 days led to better survival following challenge with A. salmonicida. There was no indication of serum or mucus antibodies to A. salmonicida, but there was an increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity in the fish.

Vibrio harveyi cells (dose— 25 μmg mL−1 of total protein concentration) destroyed haematopoietic cultures of Nephrops norvegicus within 24 h of exposure. Cytopathic effects (CPE) started after 4h of exposure to the bacterial cells, with some granularity in the cytoplasm, mostly in cells in the outer periphery of the explant growth. At the end of the infection, a considerable number of nuclei remained attached to the substrate, apparently unaffected. Following exposure to ECP, initial deterioration was observed at 2 h with the presence of granularity in the cytoplasm of

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.

The mode of action of honey bee pollen (HBP) was investigated in Nile tilapia (Oreochromis niloticus) challenged with Aeromonas hydrophila. Thus, fish with an average weight of 29 ± 3 g were divided into four groups, and fed with HBP-free diet (control), and 1%, 2.5% and 4% (w/v) HBP incorporated into basal diet for 10, 20 and 30 days. Immunological, hematological, biochemical and growth parameters were measured, and sub-groups of fish were challenged with A. hydrophila via intraperitoneal injection. HBP significantly increased the growth performance parameters [body weight, length, average daily gain (ADG), specific growth rate (SGR), and feed efficiency ratio (FER)] and immunological (phagocytic activity, serum bactericidal activity and nitroblue tetrazolium assay (NBT)), hematological (hematocrit (Hct), leucocrit (Lct), the numbers of neutrophils, monocytes and lymphocytes) and biochemical parameters (serum total protein, albumin and globulin ratios). Furthermore, all treated fish exhibited significant protection against challenge with A. hydrophila, with the highest protection (93%) observed in the group fed with 2.5% (w/v) HBP for 20 and 30 days.

Fourteen species of Vibrio were screened for chitin-induced chitinase activity in culture medium. V. carchariae, V. alginolyticus 283 and V. campbellii showed high levels of activity. Screening on agar plates containing swollen chitin showed high levels of chitinase activity by the same three species, and also by V. fischeri and V. alginolyticus 284. An affinity purification procedure was developed for the chitinase from V. carchariae. The purified chitinase was active as a monomer with M(r) 63,000-66,000, and displayed activity toward polymeric chitin from acetylated chitosan or from crab shells. N-terminal sequence analysis and immunological cross-reactivity confirmed that the enzyme belongs to the group A/chiA family of bacterial chitinases.

A strain of Carnobacterium sp., isolated from the intestine of Atlantic salmon, was evaluated for potential use as a probiotic for salmonids. In vitro studies demonstrated antagonism against Aeromonas hydrophila, A. salmonicida, Flavobacterium psychrophilum, Photobacterium damselae subsp. piscicida, Streptococcus milleri, Vibrio anguillarum and V. ordalii but not towards Debaryomyces hansenii, Janthinobacterium lividum, V. alginolyticus, V. harveyi or Yersinia ruckeri. Feeding salmonids with diets containing the probiotic revealed that the isolate remained viable in the gastrointestinal tract. After reverting to feeding with control diets, the isolate was re-isolated from the intestine up to 4 and 10 days in fingerlings and fry, respectively. After feeding with the probiotic for 14 days, challenge by cohabitation indicated effectiveness at reducing disease caused by A. salmonicida, V. ordalii and Y. ruckeri but not V. anguillarum.

Out of 19 Vibrio harveyi isolates obtained from a diversity of hosts and geographical locations, 14 were pathogenic to rainbow trout, Oncorhynchus mykiss (Walbaum), and Atlantic salmon, Salmo salar L., with mortalities of up to 100% following intraperitoneal injections of 106 cells fish−1. The extracellular products (ECPs) of only five pathogenic isolates were harmful to fish. Both pathogenic and non-pathogenic cultures produced ECPs containing caseinase, gelatinase, phospholipase, lipase and haemolysins. Vibrio harveyi VIB 645, which was the most pathogenic isolate, produced ECPs with a maximal effect on salmonids from preparations obtained by using cellophane overlays on tryptone soya agar supplemented with 1% (w/v) sodium chloride with incubation at 28 °C for 24 h. This preparation contained the highest titre of haemolytic activity to Atlantic salmon (1:256) and rainbow trout (1:32) erythrocytes.

Vibrio harveyi recovered from diseased post-larval Penaeus vannamei produced a thermostable exotoxin, which was lethal to Dublin Bay prawns, Nephrops norvegicus L., when injected intramuscularly. The extracellular products (ECPs) concentrated from tryptone soya broth supplemented with 1% (w/v) sodium chloride or from cellophane overlays on marine 2216E agar with incubation at 15, 22 and 27 °C were toxic, with the lethal dose 50% of the crude ECPs estimated to be 4.4 μgprotein prawn−1. Proteolytic, haemolytic and cytotoxic activities were detected, although the occurrence and quantity of these activities were influenced by cultural conditions. The ECPs which had been heated (100 °C for 10 min) or digested with protease K produced the same pathology as crude, untreated ECPs. Western blotting demonstrated that all the ECP preparations contained low molecular weight lipopolysaccharides, which may constitute the lethal toxin of V. harveyi.

Fifty two bacterial strains, identified as Vibrio spp., were isolated from diseased plaice fry. The most numerous group comprised V. anguillarum (26/52), of which 3 isolates belonged to serogroup O2a, 16 corresponded to serogroup O18, and 7 isolates were non-typeable. All serogroup O18 isolates had identical ribotype patterns. Fourteen isolates were identified as V. splendidus biotype I (n = 11) or V. splendidus-like (n = 3). Seven isolates were V. fluvialis, representing the first isolation of this species in Denmark and the first description of V. fluvialis associated with diseased fish. All V. fluvialis isolates had identical ribotype patterns, indicating the presence of a single clone. The last 5 isolates belonged to 2 different, unidentified Vibrio species (n = 2 and 3, respectively). Although all isolates were recovered from diseased plaice fry, their exact role as pathogens for the fry is as yet uncertain. Selected isolates were tested for virulence to salmon and turbot. When injected into juvenile salmonid fish, the recorded LD50 values were higher than 10(6), indicating that their virulence was relatively low. However, virulence seemed to deteriorate upon subculturing, and therefore, the strains may have been more virulent upon primary isolation from the plaice fry.

The small subunit ribosomal RNA (SSU rRNA) encoding genes from reference strains of Aeromonas salmonicida subsp. smithia and Haemophilus piscium were amplified by polymerase chain reaction and cloned into Escherichia coli cells. Almost the entire SSU rRNA gene sequence (1505 nucleotides) from both organisms was determined. These DNA sequences were compared with those previously described from A. salmonicida subsp. salmonicida, subsp. achromogenes and subsp. masoucida. This genetic analysis revealed that A. salmonicida subsp. smithia and H. piscium showed 99.4 and 99.6% SSU rRNA gene sequence identity, respectively, with A. salmonicida subsp. salmonicida.

Potentially harmful substances-e.g. pesticides, heavy metals and hydrocarbons-are often released into the aquatic environment. When large quantities of pollutants are released there may be an immediate impact as measured by large-scale sudden mortalities of aquatic organisms, e.g. fish kills resulting from contamination of waterways with agricultural pesticides. Lower levels of discharge may result in an accumulation of the pollutants in aquatic organisms. The end results, which may occur long after the pollutants have passed through the environment, include immunosuppression, reduced metabolism, and damage to gills and epithelia. However, the link between adverse water quality and fish diseases is not proven. Alleged pollution-related diseases include epidermal papilloma, fin/tail rot, gill disease, hyperplasia, liver damage, neoplasia and ulceration. Many surveys have indicated a greater proportion of diseased fish in polluted compared to non-polluted marine sites. Yet, the value of such surveys may be questioned. Specific examples of fish diseases thought to reflect the effects of pollution include surface lesions attributed to Serratia plymuthica, fin and tail rot caused by Aeromonas hydrophila and Pseudomonas fluorescens, gill disease resulting from the actvity of Flavobacterium spp., vibriosis as caused by Vibrio anguillarum, and enteric redmouth (causal agent, Yersinia ruckeri). Research indicated that some of the diseases caused by Aeromonas, Flavobacterium and Pseudomonas resulted from generally adverse water quality, i.e. higher than usual quantities of organic material, oxygen depletion, changes in pH values and enhanced microbial populations. Some infections with Serratia and Yersina may well have reflected contamination of waterways with domestic sewage, e.g. leaking septic tanks. At least one outbreak of vibriosis was linked to high concentrations of copper, which may have debilitated the fish making them more susceptible to disease.

Plate and dipstick enzyme-linked immunosorbent assays (ELISA) were developed for the rapid detection of Vibrio harveyi from penaeid shrimp and water. The ELISA, which incorporated a polyclonal antiserum produced in a female New Zealand white rabbit, detected 105 cells of V. harveyi/ml. Also, the systems detected V. harveyi in water from Chinese shrimp hatcheries. The systems permitted the recognition of a wide range of V.harveyi isolates, but not those of other taxa. Western blot analysis of bacterial outer membrane proteins (OMP) indicated that a wide epitope was recognised, with many immunoreactive bands in common between isolates of V. harveyi.